Field of the Invention
The present invention relates to a bone substitute and to a method for the preparation thereof.
Description of Related Art
Bone is a hybrid material constituted mainly of cells, collagen type I which constitutes an organic protein network, and a mineral phase consisting of hydroxyapatite crystals of nanometric size. This large-scale organic/mineral association in three dimensions gives the bone tissue both elasticity and hardness, allowing it to withstand the forces which are applied thereto. Bone is therefore hard, dense and very strong.
Weiner & Wagner (Ann. Rev. Mater. Sci. 28, 271-298, 1998) have proposed a description of a hierarchical organization on various scales which can be broken down into seven levels described as follows and which are illustrated by the appended FIG. 1:                level 1 (FIG. 1a): the two major constituent basic components of bone, i.e. the hydroxyapatite platelets and the striated collagen fibrils, constitute the first hierarchical level of organization. This is the lowest level of organization, on the nanometric scale. The apatite phase is in particular characterized by the presence of characteristic inter-reticular planes such as (002) and (300). The apatite crystals, at this level of organization, do not have a particular orientation. The collagen fibrils are characterized by a periodic striation, which is visible by electron microscopy, and which results from the assembly of the collagen I molecules, inducing a periodic shift of 67 nm;        level 2 (FIG. 1b): the coalignment of the hydroxyapatite platelets according to their axis c, along the main axis of the striated collagen fibrils, constitutes the second level, i.e. the inter-reticular planes (002) of the apatite are oriented perpendicular to the main axis of the fibrils and, therefore, according to the axial periodicity (i.e. according to the striations) of the collagen fibrils (striation=67 nm). The term mineralized collagen fibrils is used (width 100 to 300 nanometers). Level 2 is also on the nanometric scale;        level 3 (FIG. 1c): several mineralized collagen fibrils are assembled side by side in parallel bundles, forming a mineralized collagen fiber (width 1 to 3 micrometers). The micrometric scale is reached at organization level 3;        level 4 (FIG. 1d): it is complex since the mineralized collagen fibrils or fibers can organize in three dimensions. Specifically, at this level, it is possible to distinguish the coexistence of domains where the fibrils/fibers are aligned in a preferential direction over a large distance and/or form arched structures characteristic of their stack according to a “cholesteric” geometry. Domains where the fibrils/fibers do not organize (“isotropic” domains) are also distinguished. The scale varies from micrometric to millimetric;        level 5 (FIG. 1e): the compact bone has an arrangement of parallel cylindrical structures of millimetric size, denoted “osteons”. In section, these osteons appear to consist of concentric collagen lamellae;        level 6 (FIG. 1f): the cell-rich central part of the long bones is called “spongy bone”. In this bone, bone lamellae form a macroporous network of thin and irregular rows. The compact bone/spongy bone combination constitutes organization level 6. The scale is more than one millimeter;        level 7 (FIG. 1g): the final level is quite simply the whole bone.        
Several classes of synthetic materials (denoted “implant materials”) or natural materials (denoted “grafts”) are proposed in the prior art. Implant materials are generally bioinert, i.e. simply tolerated by the organism, or biocompatible, i.e. they integrate perfectly into the host organism. A graft is a bone tissue taken from the person for whom it is intended (autograft) or from a third person (allograft), and it is generally osteoconductive, i.e. it is capable of guiding bone regrowth.
An osteoinductive bone substitute, i.e. one which is capable of inducing bone reconstruction, nevertheless constitutes an ideal substitute. The development of such a material is complex. Putting into place such a material requires the use of constituents which have a specific crystalline phase and chemical nature in order to optimize perfect integration thereof in a human or animal body, and to thus avoid rejection. Its three-dimensional organization must be reconstituted in order to provide, firstly, the mechanical properties and, secondly, a porosity suitable for the colonization of said substitute by the host tissue. The access to the organization of the organic bone network (20% by mass), and also the association thereof with the mineral phase (70% by mass) in the tissue are very difficult to reproduce in vitro.
Many studies have been carried out with a view to synthesizing bone substitutes, and in particular studies relating to collagen mineralization. The mineralization of turkey bone tendon collagen has been studied by W. Traub et al. [Proc. Natl. Acad. Sci. USA 1989, 86, 9822-9826], but the material obtained does not display an organization analogous to that of bone. Other tests have been carried out with purified collagen in vitro, but the conditions of strong dilution under which the tests were carried out did not make it possible to obtain a material having the bone density and the three-dimensional collagen organization that are found in living bone tissues [cf. D. Lickorish et al. (J. Biomed. Mat. Res. 2004, 68A, 19-27); S. Yunoki et al. (Mat. Lett., 2006, 60, 999-1002); D. A. Wahl et al. (Eur. Cell. Mat. 2006, 11, 43-56)].
The crystallization of calcite CaCO3 from a solution of CaCl2 under an ammonia atmosphere generated by the thermal decomposition at ambient temperature of a powder of (NH4)CO3 has been described by L. Addadi et al. (Proc. Natl. Acad. Sci. USA 1987, 84, 2732-2736).
It is also known practice to precipitate collagen from an acid solution by increasing the pH. R. L. Ehrman et al. (J. Nat. Cancer Inst. 1956, 16, 1375-1403) describe a method in which a solution of collagen in acetic acid is brought into contact with NH3 vapors. It transforms into a gel containing fine grains. The structure of the material obtained is not described.
M. M. Giraud-Guille et al. (J. Mol. Biol. 1995, 251, 197-202) and (J. Mol. Biol. 1992, 224, 861-873) describe the “liquid crystal” structure obtained using a concentrated solution of collagen and also the sol-gel transition obtained by raising the pH from acidic to basic.
G. Mosser, M. M. Giraud-Guille et al. (Matrix Biol. 2006, 25, 3-13) describe a method in which an acidic solution of collagen (5 mg/mL) is gradually concentrated in glass microchambers in order to obtain a far-reaching helicoidal organization of the collagen molecules and also a concentration gradient. The solution is then brought into contact with ammonia vapors, in order to form collagen fibrils and to stabilize the organization put in place in the liquid phase.
B. A. Harley et al. (Biomaterials 2006, 27, 866-874) describe the production of a structured matrix of collagen also containing a glucosaminoglycan. Collagen microfibrils are homogeneously mixed with chondroitin sulfate at 4° C. The solution is then centrifuged in a mold, ultra-rapidly frozen, freeze-dried, and then crosslinked at 105° C. under a vacuum of 50 mTorr for 24 hours. The fibrillar nature of the collagen is not described.
C. Guo et al. (Biomaterials 2007, 28, 1105-1114) describe the use of magnetic beads for aligning a solution of collagen fibrils. A collagen solution prepared in a phosphate buffer at concentrations of 2.5 mg/ml, maintained at 4° C., is brought into contact with the magnetic beads. The same samples are also prepared in the presence of cells at a final collagen concentration of 1.2 mg/ml. In both cases, the samples are placed in a magnetic field of less than 1G during the induction of fibrillogenesis produced by an increase in temperature to 37° C. A CO2 atmosphere is also used when cells are integrated into the matrix. The matrices are very loose and the fibrillar nature of the collagen is not mentioned. M. J. Olsza et al. (Calcif. Tissue Int. 2003, 72, 583-591) describe the calcification of a collagen sponge in the presence or absence of a polymer of the poly(aspartic acid) type. The collagen sponge is constituted of collagen type I obtained from bovine tendon. The mineral is calcium carbonate and not calcium phosphate, no apatite phase is therefore obtained. The presence of striated fibers is not demonstrated and the collagen fibers are not oriented. J. H. Bradt et al. (Chem. Mater. 1999, 11, 2694-2701) describe a method in which two solutions are prepared at 4° C., the first being a solution of collagen (calf dermis collagen type I) at 1 mg/mL acidified with HCl and containing CaCl2, and the second being a buffer solution containing phosphate ions. The phosphate solution is then mixed with the collagen solution, making it possible to achieve a pH of 6.8, and the whole mixture is heated to 30° C. Coprecipitation gives a mixture of phases containing calcium phosphate, hydroxyapatite and octacalcium phosphate. In addition, the collagen fibers are isolated nonoriented fibers and do not constitute a dense matrix. N. Gehrke, N. Nassif et al. (Chem. Mater. 2005, 17, 6514-6516) describe the remineralization, with calcium carbonate, in the presence or absence of a polymer of the poly(aspartic acid) type, of the organic network of previously demineralized mother-of-pearl.
None of the synthesis methods known to date makes it possible to obtain a bone substitute which reproduces level 4 of three-dimensional organization of collagen associated with a mineral phase of apatite crystals which is observed in natural bone.